Measurement of total iron binding capacity

ABSTRACT

An apparatus for use in the measurement of total iron binding capacity of blood serum or plasma comprises a container having a removable closure and containing a measured quantity of dried iron-saturating substance, which is adhered to the inner surface of the container or of the closure and a measured quantity of dried alumina. The invention further provides a simple, rapid and inexpensive method for measurement of total iron binding capacity using the apparatus.

BACKGROUND OF THE INVENTION

This invention relates to the measurement of total iron binding capacityin serum.

Iron is carried in the blood plasma by a specific carrier protein calledtransferrin. This is a protein of molecular weight 76,000-80,000, whichhas two sites each capable of binding one iron atom. The total amount oftransferrin present determines the total iron binding capacity(T.I.B.C.) of the serum.

The estimation of the T.I.B.C. is an important clinical procedure, withan established role in the diagnosis of such conditions as irondeficiency anaemia and haemochromatosis, and in the monitoring oftherapeutic procedures. In iron deficiency states, T.I.B.C. is elevated:in iron overload conditions T.I.B.C. is depressed.

Techniques which are currently used for determination of T.I.B.C. relyon the saturation of transferrin and removal of excess iron from theserum with an adsorbent such as magnesium carbonate (Ramsay, W. N. M.:Clin. Chim. Acta 2 221 (1957)) or an ion exchange resin (Peters, T.,Giovanello T. J., Apt, L. and Ross, J. R.; J. Lab. Clin. Med. 48 274(1956)). Other methods employ direct measurement of the excess ironafter saturation, and calculation of unsaturated iron-binding capacity(Williams, H. L., and Conrad, M. E.; J. Lab. Clin. Med. 67 171 (1966);O'Malley, J. A., Hassan, A., Shiley, J., and Traynor, H.; Clin.Chemistry 16 92 (1970)). The most commonly used magnesium carbonatemethod is subject to error owing to the inclusion of non-transferrinbound iron in the supernatant solution (Ramsay, W. N. M.; J. Clin.Pathol. 26 691 (1973)).

An improved method using an alumina column, which was faster and simplerthan the magnesium carbonate method while offering improved accuracy,was previously disclosed by the present inventor (Clin. Chemistry 26 156(1980)).

SUMMARY AND OBJECTS OF THE INVENTION

The object of the present invention is to provide improvements in themeasurement of T.I.B.C.

There is provided in accordance with the invention a tube closed at oneend, having a removable closure, the tube containing a measured quantityof a dried iron-saturating substance, which is adhered to the innersurface of the base of the tube, and a measured quantity of driedalumina (aluminum oxide, Al₂ O₃).

Preferably the tube and closure are made of plastics.

Preferably the neck of the plastic tube is threaded to receive aplastics screw cap. The screw cap is most preferably large enough tocontain the quantity of alumina.

Preferably the tube is made of polystyrene, most preferably oftransparent polystyrene.

Preferably the cap is made of polyethylene.

The dried iron-saturating substance is preferably a complex salt offerric iron.

The alumina is preferably basic chromatographic grade alumina, mostpreferably Brockmann grade II.

Preferably the tube is designed to fit sample carriers of automaticanalysers such that their sample probes will sample from above the leveloccupied by alumina in the tube.

According to a further aspect of the invention, there is provided amethod of performing the measurement of T.I.B.C. using the apparatusdescribed hereinabove comprising pouring alumina out of the tube intothe cap, adding a solvent, for example water, to the tube to dissolvethe iron-saturating substance, and adding the sample of serum to thetube; then after a brief period of incubation to allow the iron to bindto transferrin in the serum sample, replacing the alumina in the tube,mixing the tube to allow binding of the unbound iron to the alumina, andafter allowing the alumina to settle, measuring the iron content of thesupernatant by any suitable means, for example by automatic continuousflow analyser or by atomic absorption spectroscopy.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING

Preferred embodiments of the invention will now be described by way ofexample only with reference to the accompanying drawings in which:

FIG. 1 represents an exploded view of a free-standing sample tube havinga screw cap, containing a measured amount of alumina powder, and havinga measured amount of an iron-saturating substance bonded to the interiorsurface of the tube;

FIG. 2 represents an exploded view of free-standing sample tube as inFIG. 1 but having a slidably fitting cap; and

FIG. 3 represents an exploded view of a free-standing sample tube as inFIG. 1, but having a screw cap with a solid central projection extendingto a hollow opening, to the interior surface of which is bonded ameasured amount of an iron-saturating substance.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to one embodiment of the invention there is provided afree-standing sample tube as shown in FIG. 1 comprising a polyethylenecap 1 which is adapted to screw onto the threaded neck 2 of apolystyrene tube 3 which is closed at one end, and has a rim 4 forming abase which enables the tube to be free-standing. The tube contains ameasured amount of washed and dried chromatographic-grade alumina powder5, and a measured amount of an iron-saturating substance 6 bonded to thebase of the interior surface of the tube 3.

According to one preferred embodiment there is provided a free-standingsample tube, comprising a polyethylene cap 1 which is adapted to fitsnugly within the neck 2 of a polystyrene tube 3 which is closed at oneend, and has a rim 4 forming a base which enables the tube to befree-standing. The tube contains a measured amount of washed and driedchromatographic-grade alumina powder 5, and a measured amount of aniron-saturating substance 6 bonded to the base of the interior surfaceof the tube 3.

The polyethylene cap defines a hemispherical enclosure large enough tocontain the alumina and constructed so that the internal diameter of thecap is identical to that of the base. The cap is designed to fit intothe base such that there is a continuous, unbroken internal surface.

The tube preferably is 44 mm high (not including the cap), has anoutside diameter of 15 mm at the top and 12.5 mm at the base, and has aworking capacity of 2 ml. More preferably the tube is 40 mm high.

Preferably 0.4 g of alumina is used.

In a preferred embodiment, the iron-saturating substance is attached tothe base of the tube 3 by adding 10 ul of a solution of FeCl₃ at aconcentration of 500 mg Fe³⁺ /l in 0.1 mole/l citric acid containing0.2% sodium azide and heating the tube in an oven at 70° until dry(approximately 3 hours).

In a specific example, the test is performed as follows:

1. Invert the tube and remove the cap so that the alumina is retained inthe cap.

2. Add 1 ml. of water to the tube and wait two minutes or longer todissolve the dried iron saturating substance which is attached to thebottom of the tube.

3. Add 0.5 ml. of specimen (serum) and stand three minutes or longer toallow the iron to bind to the carrier protein transferrin.

4. Pour the alumina back into the tube and then replace the cap.

5. Place the capped tube onto a laboratory rotator and mix by constantinversion for ten minutes or longer.

6. Remove the tube from the rotator and stand 1/2 minute or longer andeither transfer a portion of the supernatant to another sample containeror use the tube itself as a sample container. (The tube may be used asthe sample container on some automatic instruments e.g. the Techniconauto-analysers commonly used in hospitals.

7. Measure the iron content of the supernatant.

According to another preferred embodiment, there is provided a freestanding sample tube as shown in FIG. 3, comprising a polyethylene cap 1which is adapted to screw onto the threaded neck 2 of a polystyrene tube3 which is closed at one end and has a rim 4 forming a base whichenables the tube to be free standing. The tube contains a measuredamount of washed and dried chromatographic-grade alumina powder 5.

The cap 1 contains a solid central projection which extends to a hollowopening, which may for example be hemispherical. A measured amount of aniron-saturating substance 6 is bonded to the interior surface of theopening.

This cap design allows the test to be performed without decanting thealumina. The central projection to which the iron-saturating substanceis bonded restricts contact of alumina with the iron-saturatingsubstance, thereby allowing iron to dissolve and bind to transferrinbefore it is bound to alumina.

The tube preferably is 44 mm high (not including the cap), has anoutside diameter of 15 mm at the top and 12.5 mm at the base and has aworking capacity of 2 ml. The central projection has an externaldiameter of 7 mm and extends 11 mm into the tube when the cap is screwedon. The hollow opening of the projection is able to contain 10 ul ofsolution.

The iron-saturating substance is attached to the interior of the hollowopening by adding 10 ul of a solution of FeCl₃ at a concentration of 500mg Fe³⁺ /l in 0.45 mole/l citric acid containing 1% glycerol to theinverted caps and heating at 70° C. in an oven until dry (approximatelythree hours). The concentration of citric acid (0.45 mole/l) is chosento promote optimum binding or iron to transferrin in the presence ofalumina. The glycerol is added to enhance bonding of the iron-saturatingsubstance to polyethylene.

Preferably 0.4 g of alumina is used.

In a specific example the test is performed as follows:

1. Remove the cap containing the iron-saturating substance.

2. To the tube containing the alumina add 1 ml of water followed by 0.5mls of serum.

3. Replace cap and proceed with steps 5 to 7 as before.

The present invention represents a considerable improvement compared tothe prior art with respect to the cost of materials, stability ofreagents, speed and convenience of carrying out the procedure, andreliability of results. The cost of materials for the present inventionis less than one third the cost for the column technique. The preparedtubes are stable for at least six months at room temperature, comparedto the stability of the stock FeCl₃ solution of the present inventor'searlier publication (Clin. Chemistry 26 156 (1980)) which was threemonths at 4° C. The working solution in that case was stable for oneweek only. The procedure of the present invention can be performed inone minute (excluding the incubation period), compared to 11/2 minutesfor the earlier procedure.

Furthermore, a considerable amount of labour is avoided by the provisionof the reagents in pre-packaged form, eliminating the need for washingand drying and weighing or dispensing of alumina and preparation anddispensing of iron solution for the column method.

Quality control for the procedure can be enhanced by the pre-testing ofbatches of alumina before addition to the tubes. This overcomes theproblem of the occasional batch of alumina which binds ironinconsistently, leading to errors and further expense.

Thus the use of the "one-tube" technique of the present inventionresults in cost savings in both materials and labour.

The procedure of the present invention is not subject to error resultingfrom lipaemic, icteric or haemolysed serum samples. Preliminary studiesindicate that the procedure is applicable to plasma samples as well asto serum samples.

It will be clearly understood that the invention in its general aspectsis not limited to the specific details referred to hereinabove.

I claim:
 1. A one-tube method for measurement of total iron bindingcapacity using a single pre-packaged tube having a removable cap andself-contained therein a measured quantity of dried iron-saturationsubstance, which is adhered to the inner surface of the tube, and ameasured quantity of dried alumina comprising the steps of(a) pouringthe measured quantity of alumina out of the tube into the cap, (b)adding a solvent to the tube to dissolve the measured quantity ofiron-saturating substance, (c) adding a sample of serum or plasmacontaining transferrin to the tube, (d) briefly incubating to allow thedissolved iron to bind to transferrin in the sample, (e) replacing themeasured quantity of alumina in the tube, (f) mixing the tube to allowbinding of the unbound iron to the alumina, (g) allowing the alumina tosettle, and (h) measuring the iron content of the resulting supernatant.2. A method according to claim 1, wherein the steps are carried out inthe following manner(a) inverting the tube and removing the cap so thatthe alumina is retained in the cap, (b) adding 1 ml. of water to thetube and waiting two minutes or longer to dissolve the dried ironsaturating substance which is attached to the bottom of the tube, (c)adding 0.5 ml. of serum or plasma and standing three minutes or longerto allow the iron to bind to the carrier protein transferrin, (d)pouring the alumina back into the tube and then replacing the cap, (e)placing the capped tube onto a laboratory rotator and mixing by constantinversion for ten minutes or longer, (f) removing the tube from therotator and standing 1/2 minute or longer and either(i) transferring aportion of the supernatant to another sample container, or (ii) usingthe tube itself as a sample container, and (g) measuring the ironcontent of the supernatant.